Recombinant adeno-associated virus(rAAV) is a promising vector system, owing to its unique features including non-pathogenicity high titer, transduction of quiescent cells and vector integration. Recent success of efficient long term in vivo transduction accompanied by vector integration has attracted substantial interests in the field of gene therapy. However, the current rAAV system is not without shortcomings. The 5 kb vector packaging constraint precludes the use of rAAV for either large genes or inclusion of regulatory elements. In addition, the host immune response to rAAV capsid proteins, such as neutralizing antibodies, may prevent repeated virus administration. New approaches have been explored, that cotransfect the ds AAV vector plasmid DNA along with the AAV Rep gene into the cells. Despite the initial high efficiency of transfection (up to 90 percent), the plasmid integration rates are dramatically lower (up to 100 fold or more) than its viral equivalent, suggesting fundamental differences between the ds vector plasmids and ss rAAV genomes. We hypothesize that ssAAV DNA is a superior integration precursor compared to dsAAV DNA plasmid. To test this hypothesis a novel AAV inverted terminal repeat substrate termed DD-ITR has been constructed. When presented as ss plasmid DNA, this substrate can be readily converted into a DNA molecule that mimics the ssAAV viral genome, thus, a linear ssDNA with two flanking inverted terminal repeats. Using non- viral delivery systems we propose that the efficient introduction of the ssAAV DNA into the appropriate target cell(s) should overcome the vector size constraint and viral capsid immunogenicity, yet holds the potential for highly efficient integration. This proposal plans to achieve the.,following specific aims: (1) to generate novel ss AAV plasmid DNA substrates using the M13 system, which mimic the ss rAAV genome. (2) to compare the integration efficiency of the artificial ssAAV substrates to that of the ds parental plasmids and rAAV viral particles. (3) To characterize the integrants derived from ss and dsAAV plasmid DNA as well as their viral equivalents.